Biochemistry of Pannexins
The role of post-translational modifications in the regulation of Panx1 and Panx2 localization and function.
To date, it has been reported that Panx1 is a conduit of physiologically important molecules such as ATP, and intracellular Ca2+ while Panx2 channel function remains unknown. Panx1 is expressed in many mammalian tissues and its amplification is prevalent in cancer cell lines (e.g. melanoma) and tumors compared to normal tissues. Panx2 has a more restricted expression and is mainly found in the central nervous system and contribute to associated pathologies such as ischemia. Interestingly, Panx1 and Panx2 expression have been found to overlap in adult rodent brain but are inversely regulated throughout development. Diverse post-translational modifications (PTMs) have been reported on the pannexin family and have been postulated as mechanisms to regulate localization and channel activity.
Overall Goal:
Our lab seeks to understand the function and regulation of pannexins by studying the role of post-translational modifications and interacting proteins in the regulation of the subcellular localization and channel activation of pannexins.
1- Study the role of N-glycosylation in Panx2 subcellular localization and its interaction with Panx1.
N-glycosylation is a posttranslational modification that occurs in the endoplasmic reticulum (ER) and is recognized to have profound effects on protein folding and trafficking of Panx1 and Panx3. We recently have validated the predicted N-glycosylation site of Panx2 and studied the effects of glycosylation on Panx2 subcellular localization and interaction with Panx1. Our results showed that N86 is the only N-glycosylation site of Panx2 and may be important for folding and trafficking of Panx2 to the plasma membrane. Furthermore, we found that although N-glycosylation is not required for the Panx2 interaction with Pannexin 1 and the un-glycosylated forms of both pannexins can readily interact, which could ultimately be a factor in regulating their localization and channel function in cells where they are co-expressed. (See Sanchez-Pupo et al., 2018)
2- To study the effects of caspase cleavage of the Panx2 on channel activation.
Panx2 is a substrate for caspase 3 and this suggests that it may be affected during apoptosis with this modification possibly playing a role in channel opening. We hypothesize that the long Panx2 C-terminus act as a gate-regulator of the channel pore. Currently we are in process of study how Panx2 channel is regulated by caspase cleavage during apoptosis and identify the sites in Panx2 in which the caspase 3 cleavage occurs. We plan to elucidate how is the regulation of the Panx2 channels and their function during neuronal cell death using the Neuro2a cell line.
3- Study the regulation of Panx1 channels by phosphorylation (or other PTMs) under pathological conditions using melanoma as a model disease.
Among all the different PTMs reported for pannexins, phosphorylation could directly impact channel properties. Previous reports indicate that Panx1 is phosphorylated in Y198, Y308 and S206 residues under pathological conditions (Lohman et al., 2015; Weilinger et al., 2016; Poornima et al., 2015). We have reported that Panx1 is highly expressed in mouse melanoma cells and mouse melanoma tumorigenesis can be attenuated by Panx1 knockdown. Furthermore, more recently we gathered evidence that there is a high expression of PANX1 in human melanoma cell lines as well as tumors biopsies from patients at all stages of melanoma progression (Freeman et al., manuscript in revision). Remarkably, melanoma displays aberrant behavior of key signaling pathways that affect cell cycle and contribute to melanoma onset and progression. We hypothesize that phosphorylation of Panx1 is increased in melanoma cells and consequently contribute to melanoma progression. We are currently assessing, at the cellular level, whether Panx1 is differentially regulated by phosphorylation (or other PTMs) in normal melanocytes and melanoma cell lines and studying Panx1 regulation by PTMs in melanoma using an inducible melanoma mouse model and patient-derived melanoma cell lines.
